DNA polymerases are enzymes which are useful in many recombinant DNA techniques, such as nucleic acid amplification by the polymerase chain reaction (“PCR”), self-sustained sequence replication (“3 SR”), and DNA sequencing. Thermostable DNA polymerases are particularly useful. Because heat does not destroy the polymerase activity, there is no need to add additional polymerase after every denaturation step.
In its catalytic cycle, the DNA polymerase-DNA complexes formed are known to undergo a rate-limiting, conformational transition from an ‘open’ to ‘closed’ state, upon binding of the ‘correct’ dNTP or ddNTP at the active site. In the ‘closed’ state, Mg2+ (or other metal ion) mediates a rapid chemical step involving nucleophilic displacement of pyrophosphate by the 3′ hydroxyl of the primer terminus. The enzyme returns to the ‘open’ state upon the release of pyrophosphate (PPi) and translocation initiates the next round of reaction. While the ternary complex (Enzyme-DNA-dNTP (or ddNTP) can form in the absence of Mg2+ (or other metal ions), it is proficient in chemical addition of nucleotide only in the presence of Mg2+ (or other metal ions). Mg2+ (or other metal ion)-deficient conditions tend to lead to non-covalent (physical) sequestration of first ‘correct’ dNTP in a tight ternary complex (Doublie et al. (15 Feb. 1999) Structure Fold. Des., 7(2):R31-5).
Pyrophosphate-based nucleic acid sequencing method (herein below referred to as pyrosequencing) is first described by Hyman (U.S. Pat. No. 4,971,903). This technique is based on the observation that pyrophosphate (PPi) can be detected by a number of assays. In a polymerase reaction, a sequencing primer is annealed to the template. If a nucleotide complements the next base in the template (i.e. next correct base 3′ of the primer sequence), it is incorporated into the growing primer chain, and PPi is released. When only one of the four nucleotides is introduced into the reaction at a time, PPi is generated only when the correct nucleotide is introduced. Thus, the production of PPi reveals the identity of the next correct base. In this way, a sequence from a template is obtained or confirmed. Additional nucleotides of the sequence are obtained by cycling of the polymerase reaction, in the presence of a single nucleotide at a time.
Pyrosequencing does not require the separation or sizing of the reaction products by such methods as electrophoresis. It is capable of being performed in a massively parallel fashion. Pyrosequencing has been used successfully for a number of applications, including in clinical microbiology. A 96 well plate format is the most widely used format (see Biotage AB website). Recently, pyrosequencing has also been used to achieve ultra-high throughput sequencing, using template carrying microbeads deposited in microfabricated picoliter-sized reaction wells. Margulies et al., Nature advance online publication; published online 31 Jul. 2005, doi: 10.1038/nature03959.
There are several methods that can be used to detect PPi. One such assay uses two enzymes, ATP-sulfurylase and luciferase, to produce a light emission. ATP-sulfurylase generates ATP at the presence of PPi and adenosine-5′-phosphosulfate. Luciferase uses the ATP to convert luciferin to oxyluciferin, emitting a photon. Nyren et al., 151 Analytical Biochemistry 504 (1985). One problem of this reaction is that Luciferase is not absolutely specific for ATP, it can also work with dATP, and to a lesser extent may catalyze reaction with other nucleoside triphosphates as well. As such, the background can be high and non-specific reactions can happen, particularly when using dATP. Another problem with pyrosequencing is its difficulty in sequencing a stretch several consecutive nucleotides with the same base. When a stretch of the same base is present in a sequence, the polymerase continues adding the complementary nucleotides until the end of the stretch is reached and the next nucleotide requires a different base. The result is production of additional molecules of PPi, and increased light emission that is related to the number of nucleotides in the stretch. For shorter stretches of the same base, the measurement of increased light emission is sensitive enough to distinguish the length of the stretch but the accuracy of the estimate decreases as the length of the stretch increases.
Despite recent successes, the limitations of pyrosequencing remain. The current invention improves the pyrosequencing technology by overcoming these limitations.